Advancing Citrus Breeding: Next- Genereation Tools for Resistance, Flavor and Health

Abstract

New plant breeding techniques are revolutionizing citrus improvement by accelerating trait enhancement and genetic gains. In recent years, technological advances have enabled more precise and accelerated breeding. This review discusses the state-of-the-art breeding technologies for citrus, including marker-assisted selection, genomic selection, genome editing (particularly CRISPR/Cas), somatic hybridization, mutation breeding, and speed breeding. Emphasis is placed on their practical application, current limitations, and potential integration into citrus-improvement programs to address biotic and abiotic stresses, improve fruit quality, and promote sustainable production.

1. Introduction

Citrus, one of the most important fruit crops worldwide, holds a central place in global agriculture and the fresh fruit market due to its nutritional value, economic importance, and diverse applications. Citrus species such as sweet orange (Citrus sinensis), mandarin (Citrus reticulata), lemon (Citrus limon), pomelo (Citrus maxima), and grapefruit (Citrus paradisi) contribute extensively to fruit production across tropical and subtropical regions. However, despite their prominence, genetic improvements in citrus have historically been slow and complex, hindered by several intrinsic biological constraints [1].

Key limitations in traditional citrus breeding include a prolonged juvenile phase often lasting 5 to 10 years, high heterozygosity, which complicates trait fixation, and reproductive barriers such as apomixis (asexual seed formation), self-incompatibility, and polyembryony. These biological traits, combined with limited genetic diversity in many commercially cultivated varieties, reduce the efficiency of conventional breeding strategies. Moreover, the reliance on phenotype selection further delays cultivar development, often requiring several cycles of field evaluation to confirm trait inheritance and performance [2].

In recent decades, the need to address emerging challenges such as the spread of the devastating diseases Huanglongbing (HLB), citrus tristeza virus (CTV), and Phytophthora-induced root rots, as well as the pressures of climate change, water scarcity, and consumer demand for high-quality, seedless fruit has driven the citrus research community to adopt and refine innovative breeding approaches. These include advanced molecular tools and biotechnology-based techniques that allow for faster, more precise, and targeted crop improvement. New plant breeding techniques have emerged as transformative tools for overcoming traditional breeding bottlenecks in citrus. Among these, marker-assisted selection (MAS), genomic selection (GS), genome-editing technologies such as CRISPR/Cas systems, somatic hybridization (SH), mutation breeding, and speed breeding offer novel avenues to accelerate the development of elite cultivars. These technologies not only enhance the efficiency of breeding programs but also enable the integration of multiple traits such as disease resistance, abiotic stress tolerance, improved fruit quality, and seedlessness into new varieties. Furthermore, their integration with high-throughput ‘omics’ platforms, genomics, transcriptomics, proteomics, metabolomics, and epigenomics provides a deeper understanding of gene function and the regulatory networks underpinning key agronomic traits.

This review provides a comprehensive overview of the latest technological advances in citrus breeding, highlighting the mechanisms, applications, successes, and limitations of each approach. The discussion also explores how the convergence of molecular breeding and systems biology can redefine the future of citrus improvement by enabling trait stacking, precision breeding, and accelerated cultivar development. By presenting the state of the art and identifying areas of opportunity and constraint, this review aims to guide researchers, breeders, and policymakers in adopting effective strategies for sustainable citrus production in a rapidly changing agricultural landscape.

2. Molecular Breeding Technologies

2.1. Marker-Assisted Selection Breeding (MAS)

MAS involves using molecular markers linked to desired traits, disease resistance, drought tolerance, improved nutritional content, seedlessness, and fruit-quality attributes such as sugar content and acidity to accelerate selection in breeding programs [1,2]. Recent developments in high-throughput single-nucleotide polymorphism (SNP) genotyping and quantitative trait locus (QTL) mapping have allowed the identification of markers for complex traits, such as resistance to CTV [3] and tolerance to HLB [4]. One specific molecular marker, SNP08, has been identified as a reliable tool for MAS in citrus-breeding programs for resistance to Alternaria brown spot (ABS). This SNP marker, located on chromosome 3, is closely linked to the ABS resistance locus and can help distinguish between resistant and susceptible citrus genotypes [5]. The use of SNP markers enabled the identification of resistance loci for canker resistance in trifoliate hybrids [6]. In citrus, MAS is also a powerful breeding technique that accelerates the development of new citrus cultivars by using molecular markers linked to desirable traits, rather than relying solely on phenotypic evaluation [7]. Steps include trait discovery, marker discovery and validation, marker-assisted breeding, and field evaluation (Figure 1). This approach increases the efficiency and precision of citrus breeding. MAS allows plant breeders to screen for traits using DNA markers rather than waiting for plants to mature. This is particularly useful in perennial crops, such as citrus, which have a long juvenile phase [7]. Advantages of MAS in citrus include early selection before field trials, better management of polygenic traits, and efficient stacking of resistance genes. Early selection reduces breeding-cycle length and cuts down on the cost and labor needed for phenotypic screening; indeed, the method’s improved accuracy enables selection of traits with complex inheritance that are hard to measure phenotypically. MAS further enables pyramiding, whereby multiple resistance genes can be stacked in a single cultivar [8]. Trait identification determines economically important traits (Table 1).

QTL mapping uses biparental populations or genome-wide association studies (GWAS) to locate markers linked to traits. GWAS in citrus has become a powerful tool for unraveling the genetic basis of complex traits such as fruit quality, disease resistance, and stress tolerance. Unlike traditional QTL mapping, which requires biparental populations, GWAS leverages natural diversity in large citrus germplasm collections to identify trait–marker associations at high resolution [9]. Marker validation confirms the marker–trait associations across different genetic backgrounds. Markers are used to screen seedlings during breeding, enabling the selection of those with the desired alleles. Different types of molecular markers are used in citrus MAS: simple sequence repeats (SSRs) are widely used for their high polymorphism and reproducibility; SNPs are becoming increasingly popular due to their abundance and potential for high-throughput screening [10,11]. Markers for citrus diseases such as CTV, Phytophthora tolerance [12], or nematode resistance [13] are linked to resistance genes (e.g., from Poncirus trifoliata = Citrus trifoliata) and help breeders screen rootstocks early in the breeding cycle for resistant progeny [14,15]. Another example is Phytophthora root rot: resistance traits have been identified and are now traced using MAS. QTLs for resistance have also been identified in intergeneric hybrids [12,16]. Seedlessness is a key commercial trait because seeded citrus fruit has become less desirable in the United States and European markets. Seedlessness can be tracked using markers associated with parthenocarpy and sterility genes. Parthenocarpy-related markers have been applied in seedless citrus-breeding programs, especially in mandarins [17,18,19]. Fruit-quality traits such as sugar content (°Brix), acidity, aroma, and skin texture are now being mapped to specific QTLs, enabling early selection using MAS [20,21].

Table 1. Summary of MAS markers for key traits in Citrus.

Marker	Associated Trait	Resistance Type	Reference
SNP08	Alternaria brown spot	Disease resistance	[5]
SSR markers	Citrus tristeza virus, Phytophthora	Disease resistance	[12]
SNPs (various)	Seedlessness, parthenocarpy	Reproductive trait	[17,18,19]
QTL markers	Fruit-quality traits (°Brix, acidity)	Fruit-quality trait	[20,21]

Table 1. Summary of MAS markers for key traits in Citrus.

Marker	Associated Trait	Resistance Type	Reference
SNP08	Alternaria brown spot	Disease resistance	[5]
SSR markers	Citrus tristeza virus, Phytophthora	Disease resistance	[12]
SNPs (various)	Seedlessness, parthenocarpy	Reproductive trait	[17,18,19]
QTL markers	Fruit-quality traits (°Brix, acidity)	Fruit-quality trait	[20,21]

Abiotic stress tolerance of rootstocks to drought and salinity, which has become critical with the advent of climate change, is also being integrated into breeding pipelines through MAS [22]. On the other hand, the technique carries some limitations and challenges, such as the high initial cost of marker development and the need for high-quality and trait-linked markers.

2.2. Genomic Selection Breeding (GS)

GS utilizes genome-wide markers to predict breeding values, thereby accelerating selection cycles. GS has shown promise in perennial crops such as citrus, especially for polygenic traits, with high potential to predict fruit yield and internal quality traits using dense marker panels. The main advantage of GS is that it reduces breeding-cycle time and allows the selection of traits with low heritability [23,24]. GS is an emerging breeding approach in citrus that leverages whole-genome molecular markers to predict the genetic potential of individuals, enabling faster and more accurate selection. Phenotype and genotype data from a training population are used to develop statistical models (e.g., GBLUP, Bayesian, machine learning), which are trained to predict breeding values of untested individuals, enabling selection of the best candidates. High-performing genotypes are advanced before phenotyping or grafting for efficient selection. Unlike MAS, which targets specific markers linked to known traits, GS uses genome-wide data to predict complex traits that are influenced by many genes with small effects [23,25,26,27]. Citrus species present several challenges to traditional breeding: a long juvenile period; heterozygosity and apomixis, resulting in high genetic variability and asexual reproduction, respectively; and economically important traits (yield, flavor, and tolerance to stresses) being governed by multiple genes. GS can overcome these challenges. GS enables early selection based on DNA, significantly shortening the breeding cycle. Quantitative and ideal GS applications in citrus include fruit-quality improvement, in traits such as °Brix (sugar content), acidity, juice yield, and volatile compounds for aroma. Genomic prediction models are built using historical phenotypic and genotypic data from reference populations [23,24,25,28]. Climate resilience involves traits such as drought tolerance, salinity resistance, and cold hardiness, which are highly polygenic. GS models can handle these complex traits better than traditional QTL mapping or MAS [29,30]. GS is especially powerful for low-heritability traits such as disease tolerance and resistance. GS can integrate resistance to HLB, a major global threat to citrus, by uncovering real genetic targets involved in phloem regeneration and pathogen response [31]. Other pathogens, such as CTV, leprosis, Phytophthora spp., and citrus canker have been explored [32]. It can combine multiple resistance traits, even when individual gene effects are small [32].

2.3. Genome Editing Breeding

Genome editing is a transformative approach in citrus biotechnology, offering precise, efficient, and targeted modifications of the citrus genome. This technique is reshaping how researchers and breeders address challenges such as disease resistance, fruit quality, and climate resilience. The CRISPR/Cas system has revolutionized plant breeding, enabling highly precise targeted mutations (

Table 2. Examples of genome-editing targets and their outcomes.

Genome-Editing Target	Gene Edited	Improved Trait	References
LOB1 promoter	CRISPR/Cas9	Canker resistance	[34]
WRKY22	CRISPR/Cas9	Canker susceptibility	[35]
NPR3	CRISPR/Cas9	HLB tolerance	[37]
CitSWEET	CRISPR/Cas9	Sugar accumulation (sweetness)	[38]
ALS	Base editing	Herbicide resistance	[40]

). CRISPR has been successfully applied in citrus to enhance canker resistance by disrupting the effector-binding element in the promoter of the susceptibility gene LOB1 [33,34] and the transcription factor WRKY22. Mutated Wanjincheng orange plants displayed a significant resistance to canker [35,36]. Plants edited by CRISPR in NPR3, a negative regulator of systemic acquired resistance in sweet orange trees, showed improved vigor under Candidatus Liberibacter asiaticus infection (causing HLB), and delayed decline [37]. Potential fruit-quality targets include genes controlling sweetness and acidity (e.g., CitSWEET), or β-cyclase to enhance lycopene/anthocyanin content in fruit [38]. Field applications for seedlessness are limited, but laboratory-scale editing has shown promise [39]. Herbicide resistance to chlorsulfuron was acquired by editing the acetolactate synthase gene (ALS) [40]. Although genome editing refers to the use of engineered nucleases, particularly CRISPR/Cas9, there are other methods, such as transcription activator-like effector nucleases with high specificity, but these are more complex to construct. Zinc-finger nucleases are used for the introduction of targeted modifications such as insertions, deletions, or replacements in the DNA of an organism; this is labor-intensive and less versatile than other methods. CRISPR/Cas9 is the most widely used technology due to its ease of design, high efficiency, and cost-effectiveness. In citrus, editing is often conducted through Agrobacterium-mediated transformation, protoplast transfection, or particle bombardment [41]. However, improvements in genome editing raise regulatory and ethical considerations in the face of opposition raised by public opinion and environmental movements. Genome-edited citrus lines without foreign DNA (transgene-free) may bypass genetically modified organism (GMO) regulations, and sources of regulatory and public perception [42,43,44,45]. Some countries, including the United States and Japan, have relaxed their regulations for DNA-free genome editing, while the European Union still classifies these plants as GMOs [46]. Recent work [47] has demonstrated the production of transgene-free, canker-resistant citrus using Cas12a ribonucleoprotein delivery. These genome-edited citrus lines received non-GMO status from the United States Department of Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS), and Environmental Protection Agency (EPA). This same system was used to develop HLB-resistant trees, as reported by Citrus Industry Magazine [48] as an improved non-GMO citrus (Table 2). Additional future directions in the area of genome editing are multiplex genome editing, in which multiple genes are edited in one go, to pyramid traits such as flavor, resistance, and yield. Not only CRISPR/Cas9, but also the variants CRISPR/Cas12a and CRISPR/Cas13 offer new possibilities for DNA and RNA editing in citrus, such as epigenome editing, which targets gene expression without altering DNA sequences (e.g., dCas9 fusions) by adding or deleting methylation. Recently, the period for obtaining products of CRISPR in citrus has been shortened by in planta genome editing, aimed at overcoming the limitations of traditional tissue-culture methods [49].

2.4. Somatic Hybridization Breeding (SH)

SH, via protoplast fusion, allows combining genomes of sexually incompatible citrus species. This technique has been used to transfer disease resistance from wild species into commercial scions and rootstocks [50]. SH involves the fusion of protoplasts (plant cells without cell walls) from two different citrus genotypes. This creates a somatic hybrid that combines the nuclear and cytoplasmic genomes of both parents. Unlike sexual hybridization, this method allows combining genomes from sexually incompatible species or genera, such as Citrus and Poncirus. The main techniques used for SH are protoplast isolation from embryogenic callus or leaves and polyethylene glycol-mediated fusion or electrofusion. Regeneration of hybrid plants is achieved via somatic embryogenesis or organogenesis. Successful hybridization is confirmed by flow cytometry, SSR markers, and chloroplast/mitochondrial DNA analysis [51]. The fusion can be symmetric (both nuclear genomes retained), asymmetric (partial genome contribution), or cybridic (only one nuclear genome with a mixture of cytoplasmic elements) [51,52]. Cytoplasmic male sterility (CMS) and cytoplasmic hybrids are created by SH. SH is used for the study of mitochondrial or chloroplastic inheritance and to develop CMS lines for hybrid seed production [53]. SH has been used to develop disease-resistant hybrids. For example, somatic hybrids of sweet orange with lemon show resistance to Phytophthora; SH of sour orange with ‘Rangpor’ lime, Volkameriana or ‘Carrizo’ citrange resulted in hybrids demonstrating resistance to quick decline caused by CTV; fusion between Citrus sinensis and Poncirus trifoliata generated rootstocks resistant to CTV and Phytophthora [54]; SH of Fortunella, Citropsis, Atalantia, Microcitrus, and Citrus ichangensis produced hybrids that were much more resistant to the migratory endoparasitic nematode Radopholus citrophilus [55]; hybridization of Australian finger lime with sweet orange or ‘Page’ tangelo generated trees which remained HLB-negative, even after 6 years of growth in an HLB endemic environment [56]. Cytoplasmic male sterility can be induced using this method via asymmetric fusion. Somatic hybrids can be used as tetraploid parents in crosses to produce triploid (seedless) progeny. This is essential for commercial varieties such as seedless mandarins and oranges [57]. The benefits of SH in citrus lie in the ability to combine nuclear and cytoplasmic genomes from divergent species, thus bypassing sexual barriers such as sterility or seed abortion, and resulting in the development of novel rootstocks and elite scions with desired combinations of traits. As with all other methods, SH has its share of challenges and limitations, including low regeneration efficiency in some genotypes and somaclonal variation due to tissue-culture processes, and complex ploidy outcomes require screening and validation [58]. Future prospects for this method are its integration with CRISPR genome editing for precise modification of somatic hybrids; combining SH with GS and omics approaches; and developing polyploid breeding programs using somatic hybrids as intermediate materials.

2.5. Mutation Breeding and TILLING

Mutation breeding and targeting induced local lesions in genomes (TILLING) are complementary approaches used to generate and identify novel genetic variants that can be utilized for citrus improvement. These methods are especially useful in crops such as citrus, where traditional breeding is slow due to long generation times and reproductive barriers. Mutation breeding involves the use of physical (e.g., gamma rays, X-rays) or chemical (e.g., ethyl methanesulfonate) mutagens to create heritable genetic changes in plants. These mutations can result in beneficial traits, such as seedlessness [17], dwarfism, disease resistance, altered fruit quality [59], and induction of parthenocarpy and embryo abortion to create seedless mandarins [60]. Improved flavor, color, and ripening time have also been achieved. Gamma irradiation has been used to induce compact/dwarf phenotypes, which are desirable for high-density planting [61,62]. Mutation breeding has been used experimentally to induce tolerance or resistance to diseases such as Mal secco, canker, and HLB, although this application is still in the exploratory stages. In the case of Mal secco, the seedless lemon variety Ayelet, the outcome of gamma-irradiation of ‘Vilafranka’ lemon has shown tolerance in the field to Plenodomus tracheiphilus the fungal pathogen causing Mal secco disease in citrus. Trees of ‘Ayelet’ growing under disease pressure next to susceptible ‘Vilafranka’ displayed mild or no symptoms, while the mother variety, Vilafranka, showed disease symptoms within the first year of planting, with destruction of the entire orchard within a few years. In another example, Gulsen et al. (2007) described the generation of two seedless, Mal secco-tolerant varieties by irradiation of budsticks of ‘Kutdiken’ lemon with cobalt gamma irradiation [63].

TILLING is a reverse-genetics technique that enables the discovery of mutations in specific genes within a mutagenized population. It combines traditional mutation breeding with high-throughput molecular screening. It does not require transgenesis, making it more acceptable in the context of regulatory frameworks. TILLING identifies point mutations or small indels through PCR amplification and mismatch-detection methods [64]. TILLING is useful for dissecting gene function in citrus, where transgenic approaches are slow or heavily regulated. Examples for the use of TILLING in the discovery of functional properties are genes involved in fruit acidity (e.g., CitPH), peel pigmentation, or disease susceptibility [65].

A summary of these technologies with reference to their applications, trait type, and the time required to execute them is presented in

Table 3. Comparison of various parameters of the NBT.

Technology	Application	Trait Types	Time Required	Challenges
MAS	Trait-linked marker Selection	Simple, known traits	Moderate	Requires prior knowledge of QTLs
GS	Genome-wide trait Prediction	Complex/polygenic traits	Shorter breeding cycle	Requires a large training set and statistical models
Genome editing	Precise gene targeting	Biotic/abiotic traits	Laboratory-to-field time	Regulatory and delivery hurdles
SH	Whole-genome Combinations	Disease resistance, cytoplasmic male sterility	Long	Somaclonal variation, regeneration
TILLING	No transgene involved	Limited to induced mutations; screening throughput	Medium	disease resistance

.

2.6. Speed Breeding and Early Flowering Techniques

Reducing the long juvenile period is critical in citrus breeding. Techniques such as transgenic expression of Flowering locus T (FT) genes and grafting onto mature rootstocks have enabled early flowering [66,67]. Virus-induced flowering using CTV vectors to induce early bloom [68] and manipulation by controlled environmental conditions, including extended photoperiod and optimal temperature, can encourage early growth and flower bud differentiation. While less impactful than FT overexpression, this method contributes to faster vegetative development [69]. Genes involved in flowering (such as CiFT) have been modified to reduce juvenility [70].

3. Integration with ‘Omics’ Technologies

‘Omics’ technologies refer to high-throughput, holistic tools that analyze biological molecules across entire systems. Several levels of analysis are included in the concept of “omics”. Genomics provides information at the DNA level (e.g., genome sequencing, SNP discovery). Transcriptomics explores gene-expression profiles (e.g., RNA sequencing). Proteomics is the study of proteins and their modifications. Metabolomics provides a comprehensive profiling of metabolites and biochemicals, and epigenomics tracks heritable changes in gene regulation (e.g., methylation). Integration of these datasets enables multidimensional trait analysis, which is essential for citrus breeding. Reference genomes are now available for Citrus sinensis and Citrus clementina, among other species, enabling GWAS and GS [25,71].

Genomics, transcriptomics, proteomics, and metabolomics provide deep insights into gene-trait associations. These tools aid in identifying candidate genes for tolerance to HLB, drought, salinity, and other stresses. Next-generation sequencing (NGS) has transformed citrus research, enabling high-throughput genomic and transcriptomic studies that support breeding, disease resistance, and an understanding of citrus evolution. Applications of NGS in citrus research, including genome sequencing and assembly and whole-genome sequencing, have helped decode the complex hybrid origin of cultivated citrus species. For example, Citrus sinensis genome sequencing revealed interspecific hybridization between Citrus maxima (pomelo) and Citrus reticulata (mandarin) [72].

Genotyping and Marker Discovery: SNPs from resequencing are used for linkage mapping, QTL mapping, and MAS. Tools such as GBS (Ggenotyping-by-sequencing and restriction-site-associated DNA sequencing are widely used in citrus genomic studies. Use of omics technologies enabled the identification and analysis of a citrus 2OGD multi-gene locus specializing in tissue-specific furanocoumarin biosynthesis. Elimination of this gene could lead to resolving grapefruit–anti-cholesterol drug interactions [73]. Transcriptomics (RNA sequencing) is used to identify genes related to disease responses (e.g., HLB, citrus canker), ripening fruit flavor development, and drought/salinity stress [1,74,75]. Post-translational modifications and pathway regulation are analyzed by using proteomics. Proteomics was also used to identify HLB-responsive proteins in phloem tissue by correlating expression data with actual protein activity [76]. Metabolomics offers valuable insights into metabolic pathways that contribute to species differentiation and the accumulation of bioactive compounds. In citrus, metabolomics has been instrumental in identifying key genetic factors influencing flavonoid and coumarin biosynthesis. It can reveal biochemical pathways related to flavor/aroma (e.g., terpenes, flavonoids, limonoids), and disease resistance (e.g., secondary metabolites such as coumarins). It can also be useful for fruit-quality assessment and stress responses [77,78]. Epigenomics provides insight into somaclonal variation, fruit development, and stress memory and tolerance by DNA-methylation and histone-modification studies [79,80]. Some examples of how these technologies can be used for citrus improvement were recently reported: Hong et al., in their work [81], describe the construction of telomere-to-telomere (T2T) reference genomes for the cold-resistant sweet orange mutant “Longhuihong” (Citrus sinensis [L.] Osb. cv. LHH) and its wild-type counterpart “Newhall” (C. sinensis [L.] Osb. cv. Newhall). By using a multi-platform sequencing approach (PacBio HiFi, ONT, Hi-C, Illumina), this study provided valuable insights into the genetic basis of cold-stress tolerance, paving the way for the molecular breeding of frost-resistant cultivars [81]. Another interesting example comes from Guan et al. [82], who used an integrated transcriptome-metabolome analysis to reveal candidate genes involved in flavonoid biosynthesis. This research strengthened the links between gene regulation and metabolite accumulation in Citrus reticulata ‘Shiyue Ju’ (STJ) and its mutant, C. reticulata ‘Denglong Ju’ (DLJ), offering new insights into how to enhance citrus fruit quality by establishing a molecular foundation for the biosynthesis of flavonoids [82]. Furthering this approach, a broad study by Liang et al. [83] used 299 citrus accessions to link 19,829 SNPs to 653 metabolites. This research uncovered significant variation in secondary metabolites across ancestral citrus lineages, which was directly tied to divergent haplotype distributions and gene expression (e.g., flavone synthases, cytochrome P450s, prenyltransferases). Critically, it also established associations between specific metabolites and antioxidant or anticancer properties [83]. Beyond quality traits, omics technologies are also used to study central regulatory genes involved in disease resistance. For example, ERF9 (ethylene-responsive transcription factor) and TrxR1 (thioredoxin reductase) were investigated for their roles against Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing (HLB). Using WGCNA, PPI network analysis, and expression profiling, researchers identified potential molecular targets for genetic resistance and novel disease management strategies [84].

This growing body of multi-omics data, which combines genomics, transcriptomics, metabolomics, and proteomics, is proving essential for linking genotype to phenotype. These advances in omics technologies enable the targeted improvement of complex traits such as cold tolerance, nutritional quality (flavonoids), flavor, and disease resistance. By leveraging tools like marker-assisted selection, genome editing, and metabolic engineering, the citrus industry can develop cultivars that are optimized for climate resilience, consumer preference, and human health benefits.

4. Future Prospects and Challenges

Although the past decade has seen unprecedented progress in citrus biotechnology, several critical challenges remain. Most notably, the practical deployment of innovative breeding technologies is still hindered by a lack of harmonized global regulatory frameworks. While some countries (e.g., United States, Japan) permit the use of gene-edited, transgene-free crops under relaxed regulatory oversight, others (e.g., the European Union) maintain stringent GMO classifications, delaying commercialization. This regulatory inconsistency restricts the international adoption of genome editing and limits its application in breeding programs targeting global markets. Furthermore, public acceptance of genetically edited citrus remains limited. Consumer concerns regarding the safety and ethics of genome-edited food, especially in regions with historically low GMO acceptance, are often amplified by inconsistent communication strategies. This underscores a critical need for transparent, science-based public engagement to build trust and mitigate misinformation. From a scientific perspective, although integrated breeding pipelines combining tools such as CRISPR/Cas, GS, and omics-based trait discovery show great potential, their full integration into field-level citrus improvement remains complex. A significant bottleneck lies in the incomplete understanding of polygenic traits such as flavor, HLB tolerance, and drought resilience. Conflicting data on gene-expression patterns and metabolic responses across genotypes under varied stress conditions further complicate trait selection. For instance, transcriptomic analyses related to HLB response sometimes reveal inconsistent expression of key regulatory genes such as NPR3 or WRKY22 across cultivars or environmental settings.

Future research should prioritize:

• Standardizing phenotyping protocols across environments to reduce inconsistencies in genomic prediction.
• Deepening the functional validation of candidate genes identified via omics approaches.
• Developing non-GMO, transgene-free editing pipelines amenable to global commercialization.
• Expanding multilocation, multiyear field trials to evaluate the stability of edited or selected traits.

In addition, integrating ethical, legal, and socioeconomic analyses into breeding programs is crucial for guiding responsible innovation. Addressing these multidisciplinary gaps will determine whether the promise of modern citrus biotechnology can translate into sustainable, globally accepted agricultural solutions (

Table 4. Summary of regulatory status of citrus-breeding technologies (United States).

Technology	Regulatory Status	Notes
Transgenic (GMO)	Strict regulation	GMO-labeling required
CRISPR/Cas (DNA-free)	Non-GMO status (USDA/EPA)	Looser regulation if transgene-free
Mutation Breeding	Exempt	Traditional method

).

A major challenge facing the citrus industry, especially in citrus breeding, is that developing new and improved varieties is a long and difficult process. Plant breeders face a number of major obstacles that hinder progress and make it difficult to create varieties that meet the changing demands of farmers and consumers. Citrus trees grow slowly, taking many years to reach maturity and bear fruit [7]. This long waiting period delays breeding cycles and significantly slows the time it takes to release new varieties to the market. The genome of citrus is complex and varies greatly from variety to variety, making it difficult to identify desired traits [3]. Natural barriers such as self-incompatibility make it even more difficult to create varieties with specific traits [2,18]. The technological tools available today for genetic modification and genome editing have difficulty succeeding due to the low efficiency of the methods for introducing and regenerating plant tissues. The gene pool available in breeding programs is also limited, reducing the chance of finding new desirable traits and incorporating them into existing varieties. In addition, the lack of rapid and uniform methods for detecting complex traits, such as resistance to severe diseases, drought tolerance, or fruit quality, hinders the screening of new varieties with potential [see more details above].

To overcome these challenges, a combination of several innovative approaches is needed. The breeding process can be accelerated using special techniques that shorten the time it takes for the tree to reach maturity, for example, by expressing early flowering genes [66,67]. Advanced technologies such as genomic mapping can help understand the genetic complexity of citrus and enable more precise identification of the desired traits. Furthermore, innovative methods for genetic modification, such as the use of nanoparticles to introduce genetic material, can significantly increase the success rates of genome editing processes [85]. In addition, the development of automated methods for identifying and testing traits, combined with machine learning technologies, will allow for faster trait evaluation and more accurate selection of the best varieties. Expanding the gene pool through international collaborations and the integration of wild relatives of citrus can provide access to a wider range of desirable traits. Combining these strategies will allow citrus breeding programs to move from small-scale trials to large-scale implementation, ensuring that improved varieties successfully meet the demands of growers, industry, and consumers in a rapidly changing agricultural world.

5. Conclusions

Innovative breeding technologies, including CRISPR-based genome editing, GS, MAS, and omics integration, are revolutionizing citrus crops by accelerating the development of disease-resistant, climate-resilient, and high-quality cultivars. Collectively, these tools represent a paradigm shift from conventional phenotypic selection to predictive, data-driven breeding. Nevertheless, realizing the full potential of these approaches requires more than technological refinement. Strategic alignment of breeding goals with regulatory feasibility, consumer acceptance, and environmental sustainability is essential. Notably, the approval of only two genome-edited citrus trees for HLB resistance as of 2025 highlights the regulatory hurdles that still impede large-scale adoption, despite technological maturity. Therefore, future directions must focus on:

• Bridging knowledge gaps in polygenic trait architecture and gene–environment interactions.
• Creating integrated platforms for multitrait pyramiding.
• Leveraging advances in epigenome editing and multiplex CRISPR technologies to enhance efficiency.
• Fostering international collaboration to harmonize regulatory frameworks and promote public dialogue.

It is only through a combination of scientific rigor, regulatory clarity, and public engagement that innovative citrus-breeding technologies can realize their transformative impact on global citrus production.